Solid forms of treprostinil

ABSTRACT

There is provided individual polymorphic forms of treprostinil and pharmaceutical formulations comprising the same, methods of making and using the same.

RELATED APPLICATIONS

The present application is a Continuation of U.S. application Ser. No.14/200,575, filed Mar. 7, 2014, which claims priority to U.S.provisional patent application No. 61/781,303 filed Mar. 14, 2013, whichare incorporated herein by reference in their entirety.

FIELD

The present application relates in general to solid forms ofprostacyclin derivatives and in particular, to solid forms oftreprostinil and methods of making and using thereof.

SUMMARY

One embodiment is crystalline treprostinil monohydrate Form A,characterized by an X-ray powder diffractogram comprising the followingpeaks: 11.6, 16.2, and 20.0°2θ±0.2°2θ, as determined on a diffractometerusing Cu-Kα radiation at a wavelength of 1.54184 Å having a purity of atleast 90% aside from residual solvents. In another embodiment, thediffractogram further comprises peaks at 5.2, 21.7 and 27.7°2θ±0.2°2θ.In yet another embodiment, the diffractogram is substantially as shownin FIG. 2.

An additional embodiment is crystalline treprostinil monohydrate Form B,characterized by an X-ray powder diffractogram comprising the followingpeaks: 5.9, 12.1, and 24.4°2θ±0.2°2θ, as determined on a diffractometerusing Cu-Kα radiation at a wavelength of 1.54184 Å having a purity of atleast 90% aside from residual solvents. In another embodiment, thediffractogram further comprises peaks at 10.7, 20.6 and 22.3°2θ±0.2°2θ.In yet another embodiment, the diffractogram is substantially as shownin FIG. 9.

Yet another embodiment is method of making the crystalline treprostinilmonohydrate Form A comprising agitating anhydrous or wet treprostinil inan aprotic organic solvent and water followed by removal of the solventby air-drying the solid at a temperature from about 15° C. to about 35°C. until no additional solvent evaporates.

Still another embodiment is a method of making the crystallinetreprostinil monohydrate Form B comprising agitating anhydrous or wettreprostinil in a protic organic solvent and water followed by removalof the solvent by air-drying the solid at a temperature from about 15°C. to about 35° C. until no additional solvent evaporates.

One embodiment is a composition comprising substantially one form oftreprostinil monohydrate Form A or treprostinil monohydrate Form B.

In another embodiment, there is a method of treating a medicalcondition, comprising administering to a subject in need thereof apharmaceutical formulation that comprises a therapeutically effectiveamount of treprostinil monohydrate Form A or treprostinil monohydrateForm B.

In one embodiment, there is further provided a method of usingtreprostinil monohydrate form A or B in treating medical conditions,including those for which it is known in the art to use treprostinil,such as those described in Drug of the Future, 2001, 26(4), 364-374,U.S. Pat. Nos. 5,153,222; 5,234,953; 6,521,212; 6,756,033; 6,803,386;7,199,157; 6,054,486; 7,417,070; 7,384,978; 7,879,909; 8,563,614;8,252,839; 8,536,363; 8,410,169; 8,232,316; 8,609,728; 8,350,079;8,349,892; 7,999,007; 8,658,694; 8,653137; US patent applicationpublications nos. 2005/0165111; 2009/0036465; 2008/0200449;2010-0076083; 2012-0216801; 2008/0280986; 2009-0124697; 2013-0261187

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a comparison between the XRPD patterns of the uniquecrystalline XRPD patterns from solid form screen of treprostinil.

FIG. 2 is an X-ray powder diffraction pattern of treprostinilmonohydrate Form A.

FIG. 3 is a differential scanning calorimetry thermogram of treprostinilmonohydrate Form A.

FIG. 4 is a thermogravimetric thermogram of treprostinil monohydrateForm A.

FIG. 5 is a dynamic vapor sorption/desorption isotherm of Treprostinilmonohydrate Form A.

FIG. 6 is an infrared spectrum of treprostinil monohydrate Form A.

FIG. 7 is a raman spectrum of treprostinil monohydrate Form A.

FIG. 8 is a solid state ¹³C cross polarization magic angle spinningnuclear magnetic resonance spectrum of treprostinil monohydrate Form A.

FIG. 9 is an X-ray powder diffraction pattern of treprostinilmonohydrate Form B.

FIG. 10 is a differential scanning calorimetry thermogram oftreprostinil monohydrate Form B.

FIG. 11 is a thermogravimetric thermogram of treprostinil monohydrateForm B.

FIG. 12 is a dynamic vapor sorption/desorption isotherm of Treprostinilmonohydrate Form B.

FIG. 13 is an infrared spectrum of treprostinil monohydrate Form B.

FIG. 14 is a raman spectrum of treprostinil monohydrate Form B.

FIG. 15 is a solid state ¹³C cross polarization magic angle spinningnuclear magnetic resonance spectrum of treprostinil monohydrate Form B.

FIG. 16 is an X-ray powder diffraction pattern of treprostinil Form C.

FIG. 17 is a differential scanning calorimetry thermogram ofTreprostinil Form C.

FIG. 18 is an ORTEP drawing of treprostinil monohydrate Form A. Atomsare represented by 50% probability anisotropic thermal ellipsoids.

FIG. 19 is a packing diagram of treprostinil monohydrate Form A vieweddown the crystallographic a axis.

FIG. 20 is a packing diagram of treprostinil monohydrate Form A vieweddown the crystallographic b axis.

FIG. 21 is a packing diagram of treprostinil monohydrate Form A vieweddown the crystallographic c axis.

FIG. 22 is hydrogen bonded tunnels in treprostinil monohydrate Form Adown the b axis.

FIG. 23 is hydrogen bonding along the a axis in treprostinil monohydrateForm A.

FIG. 24 is an ORTEP drawing of treprostinil monohydrate Form B. Atomsare represented by 50% probability anisotropic thermal ellipsoids.

FIG. 25 v diagram of treprostinil monohydrate Form B viewed down thecrystallographic a axis.

FIG. 26 is a packing diagram of treprostinil monohydrate Form B vieweddown the crystallographic b axis.

FIG. 27 is a packing diagram of treprostinil monohydrate Form B vieweddown the crystallographic c axis.

FIG. 28 is hydrogen bonded helix down the b axis of treprostinilmonohydrate Form B.

FIG. 29 is a hydrogen-bonded tetramer that does not repeat along the baxis by hydrogen bonding between the molecules of treprostinilmonohydrate Form B.

FIG. 30 is a comparison of the two-hydrogen bonding motifs formed by thetreprostinil molecules. Water is not shown for clarity. Closed tetrameris shown on the left and open helix is shown on the right.

FIG. 31 is X-ray powder diffraction pattern of mesophase treprostinil.

FIG. 32 is X-ray powder diffraction pattern of mesophase treprostinil.

DETAILED DESCRIPTION

Unless otherwise specified, “a” or “an” means “one or more”.

Prostacyclin derivatives are useful pharmaceutical compounds possessingactivities such as platelet aggregation inhibition, gastric secretionreduction, lesion inhibition, and bronchodilation.

Treprostinil, the active ingredient in Remodulin®, Tyvaso® andOrenitram™, was first described in U.S. Pat. No. 4,306,075. Methods ofmaking treprostinil and other prostacyclin derivatives are described,for example, in Moriarty, et al in J. Org. Chem. 2004, 69, 1890-1902,Drug of the Future, 2001, 26(4), 364-374, U.S. Pat. Nos. 6,441,245,6,528,688, 6,700,025, 6,809,223, 6,756,117; 8,461,393; 8,481,782;8,242,305; 8,497,393; US patent applications nos. 2012-0190888 and2012-0197041; PCT publication no. WO2012/009816.

Various uses and/or various forms of treprostinil are disclosed, forexamples, in U.S. Pat. Nos. 5,153,222; 5,234,953; 6,521,212; 6,756,033;6,803,386; 7,199,157; 6,054,486; 7,417,070; 7,384,978; 7,879,909;8,563,614; 8,252,839; 8,536,363; 8,410,169; 8,232,316; 8,609,728;8,350,079; 8,349,892; 7,999,007; 8,658,694; 8,653137; US patentapplication publications nos. 2005/0165111; 2009/0036465; 2008/0200449;2010-0076083; 2012-0216801; 2008/0280986; 2009-0124697; 2013-0261187;PCT publication no. WO00/57701; U.S. provisional application No.61/791,015 filed Mar. 15, 2013. The teachings of the aforementionedreferences are incorporated by reference to show how to practice theembodiments of the present invention.

In sum, treprostinil is of great importance from a medicinal point ofview. Therefore, a need exists for a stable form of treprostinil, whichpresents advantage in storage, shipment, handling, and formulation, forexample.

The present invention relates to novel forms of treprostinil, includingnovel forms of treprostinil monohydrate and anhydrous treprostinil.

Treprostinil is the active ingredient of Remodulin®, which has beenapproved by the U.S. FDA for the treatment of Pulmonary ArterialHypertension (PAH) in patients with NYHA Class II, III and IV symptomsto diminish symptoms associated with exercise using subcutaneous orintravenous administration. Treprostinil is also the active ingredientin Tyvaso® inhalation solution and Orenitram™ extended-release tablets.

Treprostinil's chemical name is2-((1R,2R,3aS,9aS)-2-hydroxy-1-((S)-3-hydroxyoctyl)-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]naphthalen-5-yloxy)aceticacid of the following structure:

An anhydrous form of treprostinil has been previously described, e.g.,in J. Org. Chem. 2004, 69, 1890-1902. The anhydrous form is not stableat room temperature. Stability tests show that anhydrous treprostinil isnot stable at 25° C. and dimers formed upon standing. A larger amount ofdimers can form at higher temperatures. However, dimer formation isnegligible at 5° C. Therefore, anhydrous treprostinil must berefrigerated for storage and transport. In the past, anhydroustreprostinil had to be refrigerated and shipped with ice packs tomaintain low (2° C.-8° C.) temperatures.

The monohydrate of treprostinil has been previously described, e.g. inU.S. Pat. No. 8,350,079, the contents of which are incorporated byreference in their entirety. The monohydrate of treprostinil waspreviously described; however, the polymorphic forms of treprostinilmonohydrate and anhydrous treprostinil disclosed herein were notdescribed.

Solid Forms of Treprostinil

As described generally above, the present disclosure provides solidcrystalline forms of treprostinil in Forms A, B and Form C. Form A wassurprisingly found to be more easily filterable and easier to isolatethan the prior art compounds. The Form C may form upon drying Form A orForm B under reduced pressure and a temperature of less than 42° C.

Crystalline treprostinil monohydrate Form A is characterized by itsX-ray powder diffractogram that comprises peaks at 11.6, 16.2, and20.0°2θ±0.2°2θ, as determined on a diffractometer using Cu-Kα radiationat a wavelength of 1.54059 Å. The diffractogram comprises additionalpeaks at 5.2, 21.7 and 27.7°2θ±0.2°2θ. Form A may also be characterizedby one or more peaks in Table 1. Form A also is characterized by itsfull X-ray powder diffractogram as substantially shown in FIG. 2.

In some embodiments, Form A is characterized by its differentialscanning calorimetry (DSC) curve that comprises a minor endotherm atabout 78.3° C. and a major endotherm at about 126.3° C. Form A also ischaracterized by its full DSC curve as substantially as shown in FIG. 3.

In an embodiment, Form A is produced substantially free of any otherform of crystalline treprostinil. In another embodiment, Form A has apurity of at least 90%, 95%, 98%, 99%, or 99.9% aside from residualsolvents. Purity may be determined by a manner known in the art, such asNMR integration. It may also be determined by the lack, or reduction, ofpeaks corresponding to other forms of crystalline treprostinil in theXRPD. In one embodiment, the crystalline treprostinil monohydrate Form Ais in substantially pure form. In one embodiment, Form A is obtained inone or more of the purities disclosed above in an amount of 1 gram to 50kg. In one embodiment, the Form A is obtained in one or more of thepurities disclosed above in an amount understood by one of skill in theart to be sufficient for industrial scale production of treprostinil.

Crystalline treprostinil monohydrate Form B is characterized by itsX-ray powder diffractogram that comprises peaks at 5.9, 12.1, and24.4°2θ±0.2°2θ, as determined on a diffractometer using Cu-Kα radiationat a wavelength of 1.54059 Å. The diffractogram comprises additionalpeaks at 10.7, 20.6 and 22.3°2θ±0.2°2θ. Form B may also be characterizedby one or more peaks in Table 3. Form B also is characterized by itsfull X-ray powder diffractogram as substantially shown in FIG. 9.

In some embodiments, Form B is characterized by its differentialscanning calorimetry (DSC) curve that comprises a minor endotherm atabout 78.3° C. and a major endotherm at about 126.3° C. Form B also ischaracterized by its full DSC curve as substantially as shown in FIG.10.

In an embodiment, Form B is produced substantially free of any otherform of crystalline treprostinil. In another embodiment, Form B has apurity of at least 90%, 95%, 98%, 99%, or 99.9% aside from residualsolvents. Purity may be determined by a manner known in the art, such asNMR integration. It may also be determined by the lack, or reduction, ofpeaks corresponding to other forms of crystalline treprostinil in theXRPD. In one embodiment, the crystalline treprostinil monohydrate Form Bis in substantially pure form. In one embodiment, Form B is obtained inone or more of the purities disclosed above in an amount of 1 gram to 50kg. In one embodiment, the Form B is obtained in one or more of thepurities disclosed above in an amount understood by one of skill in theart to be sufficient for industrial scale production of treprostinil.

Anhydrous treprostinil Form C is characterized by its X-ray powderdiffractogram that comprises a peak at 6.55°2θ±0.2°2θ, as determined ona diffractometer using Cu-Kα radiation at a wavelength of 1.54059 Å.Form C may also be characterized by one or more peaks in Table 1. Form Calso is characterized by its full X-ray powder diffractogram assubstantially shown in FIG. 16.

In some embodiments, Form C is characterized by its differentialscanning calorimetry (DSC) curve that comprises a minor endotherm atabout 78.3° C. and a major endotherm at about 126.3° C. Form C also ischaracterized by its full DSC curve as substantially as shown in FIG.17.

In an embodiment, Form C is produced substantially free of any otherform of crystalline treprostinil. In another embodiment, Form C has apurity of at least 90%, 95%, 98%, 99%, or 99.9% aside from residualsolvents. Purity may be determined by a manner known in the art, such asNMR integration. It may also be determined by the lack, or reduction, ofpeaks corresponding to other forms of crystalline treprostinil in theXRPD. In one embodiment, the anhydrous treprostinil Form C is insubstantially pure form. Anhydrous treprostinil Form C differs frompolymorphic treprostinil, as can be seen by the corresponding XRPD. Inone embodiment, Form C is obtained in one or more of the puritiesdisclosed above in an amount of 1 gram to 50 kg. In one embodiment, theForm C is obtained in one or more of the purities disclosed above in anamount understood by one of skill in the art to be sufficient forindustrial scale production of treprostinil.

Mesophase treprostinil is characterized by its X-ray powderdiffractogram that comprises a lack of substantial peaks between 5.0 and40°2θ±0.2°2θ, as determined on a diffractometer using Cu-Kα radiation ata wavelength of 1.54059 Å. Mesophase treprostinil also is characterizedby one or more peaks in its X-ray powder diffractogram as substantiallyshown in any one of the X-ray powder diffractogram of FIGS. 31-32.Mesophase treprostinil also is characterized by its partial X-ray powderdiffractogram as substantially shown in any one of the X-ray powderdiffractogram of FIGS. 31-32.

In an embodiment, Mesophase treprostinil is produced substantially freeof any form of crystalline treprostinil. In another embodiment,Mesophase treprostinil has a purity of at least 90%, 95%, 98%, 99%, or99.9% aside from residual solvents. Purity may be determined by a mannerknown in the art, such as NMR integration. It may also be determined bythe lack, or reduction, of peaks corresponding to other forms ofcrystalline treprostinil in the XRPD. In one embodiment, the Mesophasetreprostinil is in substantially pure form. Mesophase treprostinildiffers from polymorphic treprostinil, as can be seen by thecorresponding XRPD. In one embodiment, Mesophase treprostinil isobtained in one or more of the purities disclosed above in an amount of1 gram to 50 kg. In one embodiment, the Mesophase treprostinil isobtained in one or more of the purities disclosed above in an amountunderstood by one of skill in the art to be sufficient for industrialscale production of treprostinil.

The mesophase treprostinil of FIG. 31 are formed, from top to bottom,from the following solutions: (1) 0.40 water activity slurry withacetone 2 days; (2) DCM slurry 60 to 30° C.; (3) 0.80 water activityslurry with isopropanol 7 days; (4) 1-propanol/water 1:1 v/v slurryambient temperature; (5) Form B+A+mesophase 50° C. 7 days, (6)Acetonitrile slurry ambient temperature; (7) Form A 50° C. 15 hours; (8)Methanol/ethyl acetate evaporation; (9) Toluene crash precipitation frommethyl ethyl ketone. Mesophase treprostinil can also be formed by thefollowing methods, as shown in FIG. 32 97% RH 12 days; mesophase+weakForm B (top) Before stress; mesophase (bottom).

Compositions and Uses of Solid Forms of Treprostinil

Another embodiment is a pharmaceutical formulation comprisingtreprostinil monohydrate Form A or Form B or anhydrous treprostinil FormC and a pharmaceutically acceptable carrier or excipient.

The term “pharmaceutical” when used herein as an adjective meanssubstantially non-deleterious to the recipient mammal. By“pharmaceutical formulation” it is meant the carrier, diluent,excipients and active ingredient(s) must be compatible with the otheringredients of the formulation, and not deleterious to the recipientthereof.

Treprostinil monohydrate Form A or Form B, or Form C, can be formulatedprior to administration. The selection of the formulation should bedecided by the attending physician taking into consideration the samefactors involved with determining the effective amount.

The total active ingredients in such formulations comprises from 0.1% to99.9% by weight of the formulation. Treprostinil monohydrate Form A orForm B, or anhydrous treprostinil Form C, can be formulated with one ormore additional active ingredients or as the sole active ingredient.

Pharmaceutical formulations of the present invention are prepared byprocedures known in the art using well known and readily availableingredients. For example, treprostinil monohydrate Form A or Form B orForm C, either alone, or in combination with other active ingredient(s)are formulated with common excipients, diluents, or carriers, and formedinto tablets, capsules, suspensions, solutions, injectables, aerosols,powders, and the like.

Pharmaceutical formulations of this invention for parenteraladministration comprise sterile aqueous or non-aqueous solutions,dispersions, suspensions, or emulsions, as well as sterile powders whichare reconstituted immediately prior to use into sterile solutions orsuspensions. Examples of suitable sterile aqueous and non-aqueouscarriers, diluents, solvents or vehicles include water, physiologicalsaline solution, ethanol, polyols (such as glycerol, propylene glycol,poly(ethylene glycol), and the like), and suitable mixtures thereof,vegetable oils (such as olive oil), and injectable organic esters suchas ethyl oleate. Proper fluidity is maintained, for example, by the useof coating materials such as lecithin, by the maintenance of properparticle size in the case of dispersions and suspensions, and by the useof surfactants.

Parenteral formulations may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents, and dispersingagents. Prevention of the action of microorganisms is ensured by theinclusion of antibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents such as sugars, sodium chloride,and the like. Injectable formulations are sterilized, for example, byfiltration through bacterial-retaining filters, or by presterilizationof the components of the mixture prior to their admixture, either at thetime of manufacture or just prior to administration (as in the exampleof a dual chamber syringe package).

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, treprostinilmonohydrate Form A or Form B, or anhydrous treprostinil Form C, is mixedwith at least one inert, pharmaceutical carrier such as sodium citrate,or dicalcium phosphate, and/or (a) fillers or extenders such asstarches, sugars including lactose and glucose, mannitol, and silicicacid, (b) binding agents such as carboxymethyl-cellulose and othercellulose derivatives, alginates, gelatin, poly(vinylpyrrolidine),sucrose and acacia, (c) humectants such as glycerol, (d) disintegratingagents such as agar-agar, calcium carbonate, sodium bicarbonate, potatoor tapioca starch, alginic acid, silicates and sodium carbonate, (e)moisturizing agents such as glycerol; (f) solution retarding agents suchas paraffin, (g) absorption accelerating agents such as quaternaryammonium compounds, (h) wetting agents such as cetyl alcohol andglycerin monostearate, (i) absorbents such as kaolin and bentonite clay,and (j) lubricants such as talc, calcium stearate, magnesium stearate,solid poly(ethylene glycols), sodium lauryl sulfate, and mixturesthereof. In the case of capsules, tablets and pills, the dosage form mayalso contain buffering agents.

Solid formulations of a similar type may also comprise the fill in softor hard gelatin capsules using excipients such as lactose as well ashigh molecular weight poly(ethylene glycols) and the like. Solid dosageforms such as tablets, dragees, capsules, pills and granules can also beprepared with coatings or shells such as enteric coatings or othercoatings well known in the pharmaceutical formulating art. The coatingsmay contain opacifying agents or agents which release the activeingredient(s) in a particular part of the digestive tract, as forexample, acid soluble coatings for release of the active ingredient(s)in the stomach, or base soluble coatings for release of the activeingredient(s) in the intestinal tract. The active ingredient(s) may alsobe microencapsulated in a sustained-release coating, with themicrocapsules being made part of a pill of capsule formulation. Use oftreprostinil monohydrate Form A or Form B, or anhydrous treprostinilForm C in solid dosage forms as tablets, dragees, capsules, pills andgranules may be preferred.

Another embodiment is a method of treating a medical conditioncomprising administering a therapeutically effective amount of theaforementioned pharmaceutical formulation, such a solid formulation,comprising the treprostinil monohydrate Form A or Form B, or anhydroustreprostinil Form C, to a subject, such as a human, in need thereof. Themedical conditions being treated include but not limited to pulmonaryhypertension (including primary and secondary pulmonary hypertension andpulmonary arterial hypertension), congestive heart failure, peripheralvascular disease, asthma, severe intermittent claudication,immunosuppression, proliferative diseases, cancer such as lung, liver,brain, pancreatic, kidney, prostate, breast, colon and head-neck cancer,ischemic lesions, neuropathic foot ulcers, and pulmonary fibrosis,kidney function, and interstitial lung disease. In some embodiments, thepharmaceutical formulation may comprise one or more active ingredientsin addition to treprostinil monohydrate Form A or Form B, or anhydroustreprostinil Form C.

Treprostinil monohydrate Form A or Form B, or anhydrous treprostinilForm C may be also used for storing, shipping and/or handlingtreprostinil.

Methods of Making

The Form A and Form B of treprostinil can be made by slurrying orprecipitating from an aqueous organic solvent. For example, an organicsolvent and greater than or equal to about 50 percent v/v water may beused. In one embodiment, the water content of the aqueous organicsolvent is from about 50 percent to about 60 or 70 or 80 percent (v/v).One of skill will understand that the slurry may comprise the waterduring part or all of the agitation, while the water in theprecipitation may be added or increased at a point to reduce thesolubility of the treprostinil and cause precipitation.

One embodiment is a method of making the crystalline treprostinilmonohydrate Form A comprising agitating anhydrous or wet treprostinil inan organic solvent and water followed by removal of the solvent byair-drying the solid at a temperature from about 15° C. to about 35° C.until no additional solvent evaporates. In one embodiment the organicsolvent is an aprotic and/or non-polar organic solvent. Examples ofnon-polar and/or aprotic organic solvents include, but not limited to,hexane, benzene, toluene, 1,4-dioxane, chloroform, diethyl ether,dicholormetane, tetrahydrofuran, ethyl acetate, acetone,dimethylformamide, acetonitrile, dimethyl sulfoxide and theircombinations. In one embodiment the organic solvent is acetone or1,4-dioxane. In one embodiment, the agitation is in the form of a slurryin 1,4-dioxane/H₂O or precipitated from acetone w/H₂O. In oneembodiment, the organic solvent is not ethanol. In one embodiment theair-drying temperature is about 15° C. to about 25° C., or anytemperature or range therein between.

One embodiment is a method of making the crystalline treprostinilmonohydrate Form B comprising agitating anhydrous or wet treprostinil inan organic solvent and water followed by removal of the solvent byair-drying the solid at a temperature from about 15° C. to about 35° C.until no additional solvent evaporates. In one embodiment the organicsolvent is a protic organic solvent. Examples of protic organic solventsinclude but not limited to formic acid, n-butanol, isopropanol,nitromehtane, methanol, acetic acid. In one embodiment the organicsolvent is methanol. In one embodiment, the agitation is in the form ofprecipitation from MeOH w/H₂O In one embodiment, the organic solvent isnot ethanol. In one embodiment the air-drying temperature is about 15°C. to about 25° C., or any temperature or range therein between.

One embodiment is a method of making the anhydrous treprostinil form Cby exposing the treprostinil monohydrate Form A and/or Form B to lowhumidity and/or vacuum at a temperature of less than 42° C. It isunderstood that the temperature must be less than 42° C., but sufficientto allow the monohydrate's water to evaporate under the appliedatmospheric pressure.

The invention will now be described in reference to the followingExamples. These examples are not to be regarded a limiting the scope ofthe present invention, but shall only serve in an illustrative manner.

EXAMPLES

Materials

Materials were used as-received and solvents were either HPLC grade orACS grade, unless stated otherwise. The treprostinil starting materialwas received cold and stored under refrigerated conditions. The solidwas generally allowed to warm to ambient temperature prior to use.Generated samples were generally stored at ambient temperature.

Preparation of Form a Monohydrate

Treprostinil (500 mg; 1.3 mmol) and 1,4-dioxane/water 1:1 v/v (3.0 mL)were charged to a glass vial. The mixture was agitated, generatinghomogeneous slurry. The slurry was left to rotate on a wheel at ambienttemperature. After approximately 3 days, the slurry was transferred tofilter paper in a laboratory fume hood to isolate the solid, spreadingthe resulting paste thin to aid in drying. Drying was continued on weighpaper, spreading the sample thin, gently breaking up and crushing thesolid as it dried. The solid, which seemed dry, was gently crushed andtransferred to a clean glass vial. The vial was covered with perforatedaluminum foil and left in a laboratory fume hood for approximately 20hours to complete drying of the solid. Weight loss during drying wasapproximately 0.9%. The white solid consisted of birefringent blades andneedles. Solid recovery was 373 mg. Experimental yield was approximately71%, accounting for 4.62% water in the solid.

TABLE 1 Observed Peaks for X-ray Powder Diffraction Pattern ofTreprostinil monohydrate Form A °2θ d space (Å) Intensity (%)  5.17 ±0.20 17.108 ± 0.689  73  5.88 ± 0.20 15.021 ± 0.528  9  7.61 ± 0.2011.624 ± 0.313  6  8.07 ± 0.20 10.952 ± 0.278  3 10.36 ± 0.20 8.537 ±0.168 91 11.62 ± 0.20 7.618 ± 0.133 58 12.59 ± 0.20 7.034 ± 0.113 3813.15 ± 0.20 6.731 ± 0.103 12 15.24 ± 0.20 5.813 ± 0.077 12 15.56 ± 0.205.695 ± 0.074 6 16.18 ± 0.20 5.479 ± 0.068 36 17.73 ± 0.20 5.002 ± 0.0572 18.17 ± 0.20 4.883 ± 0.054 20 18.77 ± 0.20 4.728 ± 0.050 7 19.00 ±0.20 4.670 ± 0.049 1 19.95 ± 0.20 4.450 ± 0.045 62 20.12 ± 0.20 4.413 ±0.044 40 20.81 ± 0.20 4.269 ± 0.041 4 21.34 ± 0.20 4.163 ± 0.039 4521.59 ± 0.20 4.116 ± 0.038 74 21.71 ± 0.20 4.094 ± 0.038 100 22.70 ±0.20 3.918 ± 0.034 51 23.06 ± 0.20 3.856 ± 0.033 32 23.82 ± 0.20 3.736 ±0.031 16 24.37 ± 0.20 3.653 ± 0.030 2 24.63 ± 0.20 3.614 ± 0.029 1824.82 ± 0.20 3.588 ± 0.029 27 25.49 ± 0.20 3.495 ± 0.027 2 26.09 ± 0.203.416 ± 0.026 21 26.49 ± 0.20 3.365 ± 0.025 2 26.87 ± 0.20 3.318 ± 0.0241 27.14 ± 0.20 3.286 ± 0.024 1 27.48 ± 0.20 3.246 ± 0.023 2 27.68 ± 0.203.223 ± 0.023 3 28.29 ± 0.20 3.154 ± 0.022 13 28.63 ± 0.20 3.118 ± 0.0215 28.88 ± 0.20 3.092 ± 0.021 3

TABLE 2 Characterization of Treprostinil monohydrate Form A MonohydrateAnalysis Result single crystal X-ray Form A structure monohydrate XRPDForm A indexed DSC endo 63° C., 49° C. onset endo 78° C. endo 119° C.endo 126° C. TGA 59° C. onset 4.4 wt % loss to 100° C. hot stage 26.6°C.; started heating 10° C./min microscopy 54.8° C.; image suggestsliquid present 62.2° C.; loss of birefringence 82.3° C.; completely lostbirefringence 121.5° C.; appeared to be crystallizing 126.6° C.;liquefaction 147.8° C.; started cooling 29.4° C.; no crystallization IRspectrum acquired Raman spectrum acquired ¹H-NMR consistent withstructure trace dioxane XRPD Form A DVS 0.7% weight gain 5 to 95% RH0.6% weight loss 95 to 5% RH XRPD Form A TG-IR TG: 4.2 wt % loss to 97°C. (25-97° C.) IR: volatile identified as water Post-TG-IR XRPDmesophase XRPD Form A KF 4.62% water ¹³C-NMR spectrum acquired

Single-Crystal Analysis of Form A

The crystals were prepared via elevated temperature (˜80° C.) slurry oftreprostinil in 1,4-dioxane/water (˜1:2.5 v/v) overnight. Crystals wereisolated from this sample in Paratone-N oil for single crystal X-raysubmission.

A colorless needle of C₂₃H₃₆O₆ [C₂₃H₃₄O₅, H₂O] having approximatedimensions of 0.20 ×0.08×0.06 mm, was mounted on a fiber in randomorientation. Preliminary examination and data collection were performedwith Cu K_(α) radiation (λ=1.54184 Å) on a Rigaku Rapid IIdiffractometer equipped with confocal optics. Refinements were performedusing SHELX97 Sheldrick, G. M. Acta Cryst., 2008, A64, 112.

Cell constants and an orientation matrix for data collection wereobtained from least-squares refinement using the setting angles of 10698reflections in the range 3°<θ<66°. The refined mosaicity fromCrystalClear is 1.25° indicating poor crystal quality. CrystalClear: AnIntegrated Program for the Collection and Processing of Area DetectorData, Rigaku Corporation, © 1997-2002. The space group was determined bythe program XPREP. Bruker, XPREP in SHELXTL v. 6.12, Bruker AXS Inc.,Madison, Wis., USA, 2002. From the systematic presence of the followingconditions: hkl h+k=2n, and from subsequent least-squares refinement,the space group was determined to be C2 (no. 5).

The data were collected to a maximum 2θ value of 133.14°, at atemperature of 150±1 K.

Frames were integrated with CrystalClear. A total of 10698 reflectionswere collected, of which 3963 were unique. Lorentz and polarizationcorrections were applied to the data. The linear absorption coefficientis 0.686 mm⁻¹ for Cu K_(α) radiation. An empirical absorption correctionusing CrystalClear was applied. Transmission coefficients ranged from0.858 to 0.960. A secondary extinction correction was applied [1]. Thefinal coefficient, refined in least-squares, was 0.00320 (in absoluteunits). Intensities of equivalent reflections were averaged. Theagreement factor for the averaging was 5.42% based on intensity.

The structure was solved using the Patterson heavy-atom method whichrevealed the position of one O atom. The remaining atoms were located insucceeding difference Fourier syntheses. Hydrogen atoms residing onoxygen atoms were refined independently. All other hydrogen atoms wereincluded in the refinement but restrained to ride on the atom to whichthey are bonded. The structure was refined in full-matrix least-squaresby minimizing the function:Σw(|F _(o)|² −|F _(c)|²)²

The weight w is defined as 1/[σ²(F_(o) ²)+(0.0478P)²+(1.5389P)], whereP=(F_(o) ²+2F_(c) ²)/3.

Scattering factors were taken from the “International Tables forCrystallography.” International Tables for Crystallography, Vol. C,Kluwer Academic Publishers: Dordrecht, The Netherlands, 1992, Tables4.2.6.8 and 6.1.1.4. Of the 3963 reflections used in the refinements,only the reflections with F_(o) ²>2σ(F_(o) ²) were used in calculatingthe fit residual, R. A total of 2595 reflections were used in thecalculation. The final cycle of refinement included 284 variableparameters and converged (largest parameter shift was <0.01 times itsestimated standard deviation) with unweighted and weighted agreementfactors of:R=Σ|F _(o) −F _(c) |/ΣF _(o)=0.056

$R_{w} = {\sqrt{\left( {\sum{{w\left( {F_{o}^{2} - F_{c}^{2}} \right)}^{2}/{\sum{w\left( F_{o}^{2} \right)}^{2}}}} \right)} = 0.119}$The standard deviation of an observation of unit weight (goodness offit) was 1.153. The highest peak in the final difference Fourier had aheight of 0.25 e/Å³. The minimum negative peak had a height of −0.26e/Å³. The Flack factor for the determination of the absolute structurerefined to 0.3(4). Flack, H. D. Acta Cryst. 1983, A39, 876.

The ORTEP diagram was prepared using the ORTEP III (Johnson, C. K.ORTEPIII, Report ORNL-6895, Oak Ridge National Laboratory, TN, U.S.A.1996. OPTEP-3 for Windows V1.05, Farrugia, L. J., J. Appl. Cryst. 1997,30, 565) program within the PLATON software package. Spek, A. L. PLATON.Molecular Graphics Program. Utrecht University, Utrecht, TheNetherlands, 2008. Spek, A. L, J. Appl. Cryst. 2003, 36, 7. Atoms arerepresented by 50% probability anisotropic thermal ellipsoids. Packingdiagrams were prepared using CAMERON modeling software. Watkin, D. J.;Prout, C. K.; Pearce, L. J. CAMERON, Chemical CrystallographyLaboratory, University of Oxford, Oxford, 1996. Assessment of chiralcenters was performed with the PLATON software package. Absoluteconfiguration is evaluated using the specification of molecularchirality rules. See, e.g., Cahn, R. S.; Ingold, C; Prelog, V. Angew.Chem. Intern. Ed. Eng., 1966, 5, 385 and Prelog, V. G. Helmchen. Angew.Chem. Intern. Ed. Eng., 1982, 21, 567. Additional figures were generatedwith the Mercury 3.0 visualization package. Macrae, C. F. Edgington, P.R. McCabe, P. Pidcock, E. Shields, G. P. Taylor, R. Towler M. and van deStreek, J.; J. Appl. Cryst., 2006, 39, 453-457. Hydrogen bonding isrepresented as dashed lines.

The monoclinic cell parameters and calculated volume are: a=30.213(5) Å,b=4.4372(6) Å, c=22.079(4) Å, α=90.00°, β=129.545(9°), γ=90.00°,V=2282.4(6) Å³. The formula weight of the asymmetric unit in the crystalstructure of treprostinil monohydrate Form A is 408.54 g mol⁻¹ with Z=4,resulting in a calculated density of 1.189 g cm⁻³. The space group wasdetermined to be C2. The space group and unit cell parameters are inagreement with those determined previously for Form A from XRPDindexing.

The quality of the structure obtained is high, as indicated by the fitresidual, R of 0.056 (5.6%). R-values in the range of 0.02 to 0.06 arequoted for the most reliably determined structures. Glusker, JennyPickworth; Trueblood, Kenneth N. Crystal Structure Analysis: A Primer,2^(nd) ed.; Oxford University press: New York, 1985; p. 87.

An ORTEP drawing of treprostinil monohydrate Form A is shown in FIG. 18.The molecule observed in the asymmetric unit of the single crystalstructure is consistent with the proposed molecular structure providedin herein. The asymmetric unit shown in FIGS. 19 to 23 contains onetreprostinil molecule for every one water molecule, indicating that FormA is a monohydrate.

The single crystal structure of treprostinil was determined to confirmthe molecular structure and the observed absolute configuration isconsistent with the proposed absolute configuration. The structure oftreprostinil was determined to be a monohydrated crystal form,designated Form A. The crystal structure contains one treprostinilmolecule and one water molecule in the asymmetric unit.

Preparation of Form B Monohydrate

Treprostinil (1019 mg; 2.6 mmol) and methanol (3.5 mL) were charged to aglass vial. The mixture was agitated and sonicated, generating a clearsolution. The solution was filtered to a clean glass vial and combinedwith water (3.5 mL), resulting in solid slurry. The vial was capped andleft at ambient temperature. After approximately 3 days, the resultingthick paste was transferred to filter paper in a laboratory fume hood toisolate the solid, spreading thin to aid in drying. Drying was continuedon weigh paper, spreading the sample thin, gently breaking up andcrushing the solid as it dried. The solid, which seemed damp, was gentlycrushed and transferred to a clean glass vial. The vial was left in alaboratory fume hood for approximately 44 hours to complete drying ofthe solid, periodically breaking up and crushing the solid to aid indrying. Drying was done with and without a perforated aluminum foilcover on the vial. Weight loss during drying was approximately 32.4%.The white solid consisted of birefringent needles in dendritic-rosetteclusters. Solid recovery was 956 mg. Experimental yield wasapproximately 82%, accounting for 12.24% water in the solid. The solidformed hard chunks during slurry and drying.

In a similar procedure to the single-crystal determination for Form A,Form B was determined.

Frames were integrated with CrystalClear. A total of 21922 reflectionswere collected, of which 7134 were unique. Lorentz and polarizationcorrections were applied to the data. The linear absorption coefficientis 0.683 mm⁻¹ for Cu K_(α) radiation. An empirical absorption correctionusing CrystalClear was applied. Transmission coefficients ranged from0.837 to 0.986. A secondary extinction correction was applied [1]. Thefinal coefficient, refined in least-squares, was 0.000370 (in absoluteunits). Intensities of equivalent reflections were averaged. Theagreement factor for the averaging was 5.76% based on intensity.

The structure was solved using the Patterson heavy-atom method whichrevealed the position of one O atom. The remaining atoms were located insucceeding difference Fourier syntheses. Some of the hydrogen atoms wererefined independently, though the majority of the hydrogen atoms wereincluded in the refinement but restrained to ride on the atom to whichthey are bonded. The structure was refined in full-matrix least-squaresby minimizing the function:Σw(|F _(o)|² −|F _(c)|²)²

The weight w is defined as 1/[σ²(F_(o) ²)+(0.0589P)²+(3.3421P)], whereP=(F_(o) ²+2F_(c) ²)/3.

Scattering factors were taken from the “International Tables forCrystallography”. Of the 7134 reflections used in the refinements, onlythe reflections with F_(o) ²>2σ(F_(o) ²) were used in calculating thefit residual, R. A total of 3905 reflections were used in thecalculation. The final cycle of refinement included 551 variableparameters and converged (largest parameter shift was <0.01 times itsestimated standard deviation) with unweighted and weighted agreementfactors of:R=Σ|F _(o) −F _(c) |/ΣF _(o)=0.068

$R_{w} = {\sqrt{\left( {\sum{{w\left( {F_{o}^{2} - F_{c}^{2}} \right)}^{2}/{\sum{w\left( F_{o}^{2} \right)}^{2}}}} \right)} = 0.135}$

The standard deviation of an observation of unit weight (goodness offit) was 1.063. The highest peak in the final difference Fourier had aheight of 0.28 e/Å³. The minimum negative peak had a height of −0.22e/Å³. The Flack factor for the determination of the absolute structurerefined to 0.0(4).

The monoclinic cell parameters and calculated volume are: a=29.8234(8)Å, b=4.63510(10) Å, c=36.126(3) Å, α=90.00°, □β=113.334(8°), γ=90.00°,V=4585.5(4) Å³. The formula weight of the asymmetric unit in the crystalstructure of treprostinil monohydrate Form B is 407.53 g mol⁻¹ with Z=8,resulting in a calculated density of 1.181 g cm⁻³. The space group wasdetermined to be C2. The space group and unit cell parameters are inagreement with those obtained previously from XRPD indexing.

The quality of the structure obtained is moderate, as indicated by thefit residual, R of 0.068 (6.8%). R-values in the range of 0.02 to 0.06are quoted for the most reliably determined structures. See, e.g.,Glusker, Jenny Pickworth; Trueblood, Kenneth N. Crystal StructureAnalysis: A Primer, 2^(nd) ed.; Oxford University press: New York, 1985;p. 87. While the overall quality of the structure falls outside of thestandard range, the data was sufficient to determine the molecularconformation of the treprostinil molecule and the contents of theasymmetric unit.

The single crystal structure of treprostinil was determined to confirmthe molecular structure and the observed absolute configuration isconsistent with that of the proposed molecular structure. The structureof treprostinil was determined to be a monohydrated crystal form,designated Form B. The crystal structure contains two treprostinilmolecules and two water molecules in the asymmetric unit. The absolutestructure was determined from the crystal structure to most likely beR,R,S,S, and S configuration at C11 (C21), C12 (C22), C14 (C24), C113(C213), and C116 (C216), respectively. All peaks in the experimentalpattern are represented in the calculated XRPD pattern, indicating thebulk material is likely a single phase.

TABLE 3 Observed Peaks for X-ray Powder Diffraction Pattern ofTreprostinil monohydrate Form B °2θ d space (Å) Intensity (%)  2.66 ±0.20 33.230 ± 2.702  3  5.32 ± 0.20 16.625 ± 0.649  53  5.92 ± 0.2014.936 ± 0.522  33  6.44 ± 0.20 13.735 ± 0.440  12  8.02 ± 0.20 11.020 ±0.281  1  9.86 ± 0.20 8.970 ± 0.185 3 10.66 ± 0.20 8.297 ± 0.158 5412.10 ± 0.20 7.314 ± 0.122 38 12.90 ± 0.20 6.861 ± 0.108 35 13.10 ± 0.206.757 ± 0.104 19 15.81 ± 0.20 5.605 ± 0.071 13 16.13 ± 0.20 5.496 ±0.069 27 16.96 ± 0.20 5.227 ± 0.062 2 17.21 ± 0.20 5.151 ± 0.060 1 17.83± 0.20 4.974 ± 0.056 3 18.07 ± 0.20 4.910 ± 0.055 1 18.52 ± 0.20 4.792 ±0.052 22 18.72 ± 0.20 4.741 ± 0.051 12 19.45 ± 0.20 4.563 ± 0.047 4819.80 ± 0.20 4.483 ± 0.045 29 20.17 ± 0.20 4.402 ± 0.044 29 20.56 ± 0.204.321 ± 0.042 65 20.99 ± 0.20 4.232 ± 0.040 12 21.22 ± 0.20 4.186 ±0.039 37 21.56 ± 0.20 4.122 ± 0.038 100 22.26 ± 0.20 3.994 ± 0.036 6222.91 ± 0.20 3.881 ± 0.034 9 23.10 ± 0.20 3.851 ± 0.033 27 23.85 ± 0.203.731 ± 0.031 7 24.38 ± 0.20 3.651 ± 0.030 53 24.60 ± 0.20 3.619 ± 0.02926 25.19 ± 0.20 3.536 ± 0.028 17 25.34 ± 0.20 3.515 ± 0.028 10 26.00 ±0.20 3.427 ± 0.026 10 26.39 ± 0.20 3.378 ± 0.025 3 26.86 ± 0.20 3.320 ±0.024 4 27.09 ± 0.20 3.292 ± 0.024 5 27.39 ± 0.20 3.256 ± 0.023 6 27.66± 0.20 3.225 ± 0.023 2 28.48 ± 0.20 3.134 ± 0.022 11 28.68 ± 0.20 3.113± 0.021 6 29.16 ± 0.20 3.062 ± 0.021 5 29.36 ± 0.20 3.042 ± 0.020 6

TABLE 4 Characterization of Treprostinil monohydrate Form B MonohydrateAnalysis Result single crystal X-ray Form B structure monohydrate XRPDForm B indexed ¹H-NMR consistent with structure XRPD Form B Ramanspectrum acquired XRPD Form B DSC endo 61° C., 48° C. onset endo 75° C.endo 118° C. endo 125° C. TGA 58° C. onset 4.4 wt % loss to 100° C. IRspectrum acquired DVS 0.3% weight gain upon equilibration at 55% RH 0.4%weight gain 55 to 95% RH 0.6% weight loss 95 to 5% RH XRPD Form B KF12.24% water ¹³C-NMR spectrum acquired

Preparation of Form C Dehydrate

Treprostinil monohydrate Form B (521 mg) was charged to a glass vial.The vial was covered with a filter and exposed to vacuum at ambienttemperature for approximately 20 hours to dry the solid. Weight lossduring drying was approximately 15.7%. The resulting solid was white andcontained 0.0% water. Solid recovery was 439 mg.

TABLE 5 Observed Peaks for XRPD of Treprostinil Form C °2θ d space (Å)Intensity (%)  3.06 ± 0.20 28.876 ± 2.019  2  4.36 ± 0.20 20.252 ±0.972  1  6.55 ± 0.20 13.490 ± 0.424  100 11.78 ± 0.20 7.511 ± 0.129 612.13 ± 0.20 7.294 ± 0.122 7 12.55 ± 0.20 7.052 ± 0.114 17 13.17 ± 0.206.723 ± 0.103 20 14.76 ± 0.20 6.003 ± 0.082 2 17.33 ± 0.20 5.117 ± 0.0597 17.98 ± 0.20 4.933 ± 0.055 16 18.27 ± 0.20 4.857 ± 0.053 9 18.74 ±0.20 4.736 ± 0.051 6 19.64 ± 0.20 4.521 ± 0.046 20 20.04 ± 0.20 4.431 ±0.044 26 20.71 ± 0.20 4.290 ± 0.041 45 21.41 ± 0.20 4.151 ± 0.039 1222.60 ± 0.20 3.935 ± 0.035 2 23.16 ± 0.20 3.840 ± 0.033 3 23.60 ± 0.203.770 ± 0.032 3 25.19 ± 0.20 3.536 ± 0.028 10 25.95 ± 0.20 3.433 ± 0.0261 27.51 ± 0.20 3.243 ± 0.023 1 29.20 ± 0.20 3.059 ± 0.021 2

TABLE 6 Characterization of Treprostinil Form C Dehydrate AnalysisResult XRPD Form C DSC endo 95° C., 90° C. onset endo 119° C. endo 126°C. TGA 0.2 wt % loss to 100° C. hot stage 25.2° C.; started heating 10°C./min microscopy 97.0° C.; change in birefringence, possiblerecrystallization 104.7° C.; started cooling 10° C./min 75.3° C.; nochange; started heating 10° C./min 115.7° C.; started heating 2° C./min119.1° C.; appeared to be growth of irregular acicular-shaped particles;started heating 10° C./min 124.1° C.; liquefaction onset IR spectrumacquired Raman spectrum acquired ¹H-NMR consistent with structure weakunknown peak at 0.07 ppm DVS 0.1% weight loss upon equilibration at 5%RH 0.2% weight gain 5 to 75% RH 4.6% weight gain 75 to 95% RH 0.5%weight loss 95 to 5% RH post-DVS XRPD B + A XRPD Form C KF 0.00% water¹³C-NMR spectrum acquired

Experimental Methods

Approximate Solubility

Solubility was estimated by a solvent addition method in which a weighedsample was treated with aliquots of the test solvent. The mixture wasgenerally vortexed and/or sonicated between additions to facilitatedissolution. Complete dissolution of the test material was determined byvisual inspection. Solubility was estimated based on the total solventused to provide complete dissolution. The actual solubility may begreater than the value calculated because of the use of solvent aliquotsthat were too large or due to a slow rate of dissolution. The solubilityis expressed as “less than” if dissolution did not occur during theexperiment.

Evaporation

Solvents were added to weighed solid in glass vials. Samples were oftenheated, agitated and/or sonicated to facilitate dissolution. Theresulting solutions were filtered into clean vials which were leftuncovered (fast evaporation) or with a loose cap (slow evaporation) toevaporate solvents in a laboratory fume hood at ambient or specifiedstirplate setpoint temperature. Solutions were also rotary evaporated.Samples were taken to dryness unless specified.

Slurry

Mixtures were generated in glass vials so that undissolved solidremained. Samples were agitated on a stirplate at specified setpointtemperature, unless indicated, or on a rotating wheel at ambienttemperature. At specified times, samples were removed for examination byPLM and/or solid recovery for XRPD analysis. Solid was generallyrecovered via vacuum filtration or paste transfer to filter paper,allowing the solid to dry in a laboratory fume hood, unless specified.

Slurries at specific water activities [7,8,9,10] were conducted usingacetone, ethanol, isopropanol, and methanol. The slurries were preparedusing aqueous solvent mixtures and/or adding water to solid, followed byspecified solvents. The slurries were sampled for XRPD at specifiedtimes, pipeting into 1.0 mm glass capillaries and concentrating thesolid via centrifugation. Prior to the first sampling, solid and/oraqueous solvent mixtures were added to some of the slurries to maintainslurry consistency. The acetone slurry at 0.8 water activity could notbe sampled directly into the capillary, so solid was isolated viadecantation of the supernatant and partially dried on filter paper in alaboratory fume hood, prior to packing in the capillary.

Slow Cool

For slow cool experiments, solutions were prepared at specifiedstirplate setpoint temperatures and filtered to clean glass vials. Theheat was shut off, allowing the samples to cool slowly to ambienttemperature. If precipitation was insufficient, samples were placedunder refrigerated conditions. Solid was isolated in the same mannerdescribed for slurry.

Crash Cool

For crash cool experiments, solutions were prepared at ambient orspecified stirplate setpoint temperature and filtered to clean glassvials. The solutions were cooled rapidly via a cold bath of dry ice andisopropanol, leaving in the bath for at least a few minutes. Ifprecipitation was insufficient, samples were placed under refrigeratedconditions. Solid was isolated in the same manner described for slurry.

Crash Precipitation

For crash precipitation experiments, solutions were filtered into glassvials containing a known volume of antisolvent, or aliquots ofantisolvent were added to the filtered solutions. If precipitation wasinsufficient, samples were left at ambient temperature or otherspecified conditions. Solid was isolated in the same manner describedfor slurry.

Vapor Diffusion

For vapor diffusion experiments, glass vials containing filteredsolutions were exposed to various vapors by placing into larger vialswith antisolvent in the bottom.

Milling

Milling was carried out in an agate jar, with agate ball, in a RetschMM200 mixer mill, using approximately 100 mg of solid. The solid wasground 6 times at 30 Hz, 2 minutes per grind, scraping the solid fromthe agate after each grind.

Melt/Quench

Solids were heated using a hot plate, Thomas-Hoover capillary meltingpoint apparatus or Wagner & Munz Heizbank system (Kofler Type WME).Heating was continued until all of the solids appeared to have melted.Rapid solidification (quench) of the melt was achieved via removal to achilled metallic heat sink or ambient-temperature laboratory bench.

The hot plate experiment was done in a glass vial. The heat setting was130 to 140° C. Solid was scraped down from the vial walls and the vialwas slowly rolled to encourage complete liquefaction of the solid.Solidification of the melt occurred quickly as vial surfaces lostcontact with the heat.

For the capillary experiment, a 1.0 mm glass capillary was placed insidea slightly larger glass capillary. Upon quench, the material spreadaround the walls of the capillary, thus the solid packing was no longerdense enough for XRPD. Temperature was measured by a NIST-traceablethermometer.

The Kofler experiment was done on a glass slide, moving the sampleacross the hot bench to pass the entire solid through approximately 141°C. The hot bench was calibrated using USP melting point standards.

Lyophilization

Solids were dissolved in 1,4-dioxane or 1,4-dioxane/water mixtures. Theresulting solutions were filtered and then frozen slowly by freezer orquickly by cold bath of dry ice and isopropanol. The frozen sample wasplaced under vacuum at approximately −50° C. using an FTSsystemsFlexi-Dry freeze dryer.

Environmental Stress

Solids were stressed in glass vials under various drying and relativehumidity (RH) environments for specified times, generally monitoringweight change during stressing. Drying was done via ambient, P₂O₅,vacuum (ambient and elevated temperatures), and convection ovenexperiments, for which the only drying condition listed in the tables isthe oven temperature. Ambient experiments were conducted by leavingsamples exposed in a laboratory fume hood. Specific RH values wereachieved by placing the sample inside sealed chambers containingsaturated salt solutions or into separate chambers containing P₂O₅powder for 0% RH. The salt solutions were selected and prepared based anASTM standard procedure. For vacuum experiments, vials were covered withnylon filters to prevent potential solid loss. For the elevatedtemperature experiments, temperature was measured by a NIST-traceablethermometer. For other stress experiments, vials were covered withperforated aluminum foil or left uncovered. Solids were stored atambient temperature in sealed vials prior to XRPD analysis.

Polarized Light Microscopy (PLM)

In general, PLM was performed using a Leica MZ12.5 stereomicroscope.Samples were viewed in situ or on a glass slide (generally covered inmineral or Paratone-N oils) with or without crossed polarizers and afirst order red compensator using various objectives ranging from0.8-10x. Crystallinity is indicated by the observance of birefringenceand extinction.

For lot 01C10010, PLM was performed using a Leica DM LP microscopeequipped with a SPOT Insight™ color digital camera. The sample wasplaced on a glass slide, a cover glass was placed over the sample, and adrop of mineral oil was added to cover the sample by capillarity. Thesample was observed using a 10, 20 and 40□ objectives with crossedpolarizers and a first order red compensator. Images were captured usingSPOT software (v. 4.5.9). A micron bar was inserted onto each image as areference for particle size.

X-ray Powder Diffraction (XRPD)

Inel XRG-3000 Diffractometer

XRPD patterns were collected with an Inel XRG-3000 diffractometer. Anincident beam of Cu Kα radiation was produced using a fine-focus tubeand a parabolically graded multilayer mirror. Prior to the analysis, asilicon specimen (NIST SRM 640d) was analyzed to verify the observedposition of the Si 111 peak is consistent with the NIST-certifiedposition. A specimen of the sample was packed into a thin-walled glasscapillary, and a beam-stop was used to minimize the background from air.Diffraction patterns were collected in transmission geometry usingWindif v. 6.6 software and a curved position-sensitive Equinox detectorwith a 2θ range of 120°. The data acquisition parameters for eachpattern are displayed above the image in Appendix C; data are displayed2.5-40°2θ.

PANalytical X'Pert PRO Diffractometer

High resolution XRPD patterns were collected with a PANalytical X'PertPRO MPD diffractometer using an incident beam of Cu radiation producedusing an Optix long, fine-focus source. An elliptically gradedmultilayer mirror was used to focus Cu Kα X-rays through the specimenand onto the detector. Prior to the analysis, a silicon specimen (NISTSRM 640d) was analyzed to verify the observed position of the Si 111peak is consistent with the NIST-certified position. A specimen of thesample was sandwiched between 3-μm-thick films and analyzed intransmission geometry. A beam-stop, short antiscatter extension andantiscatter knife edge were used to minimize the background generated byair. Soller slits for the incident and diffracted beams were used tominimize broadening from axial divergence. Diffraction patterns werecollected using a scanning position-sensitive detector (X'Celerator)located 240 mm from the specimen and Data Collector software v. 2.2b.The data acquisition parameters for each pattern are displayed above theimage in Appendix C including the divergence slit (DS) before the mirrorand the incident-beam antiscatter slit (SS); data are displayed2.5-40°2θ.

XRPD patterns were collected with a PANalytical X'Pert PRO MPDdiffractometer using an incident beam of Cu Kα radiation produced usinga long, fine-focus source and a nickel filter. The diffractometer wasconfigured using the symmetric Bragg-Brentano geometry. Prior to theanalysis, a silicon specimen (NIST SRM 640d) was analyzed to verify theobserved position of the Si 111 peak is consistent with theNIST-certified position. A specimen of the sample was prepared as a/thin, circular layer centered on a silicon zero-background substrate.Antiscatter slits (SS) were used to minimize the background generated byair. Soller slits for the incident and diffracted beams were used tominimize broadening from axial divergence. Diffraction patterns werecollected using a scanning position-sensitive detector (X'Celerator)located 240 mm from the sample and Data Collector software v. 2.2b. Thedata acquisition parameters for each pattern are:

Form A: Panalytical X-Pert Pro MPD PW3040 Pro X-ray Tube: Cu(1.54059 A)Voltage: 45 kV Amperage: 40 mA Scan Range: 1.00-39.99°2θ Step Size:0.017°2θ Collection Time: 719 s Scan Speed: 3.3°/min Slit DS: 112° SS:null Revolution Time: 1.0 s Mode: Transmission

Form B: Panalvtical X-Pert Pro MPD PW3040 Pro X-ray Tube: Cu(1.54059 A)Voltage: 45 kV Amperage: 40 mA Scan Range: 1.00-39.99°2θ Step Size:0.017°2θ Collection Time: 3883 s Scan Speed: 0.6°/min Slit: DS: 1/2° SS:null Revolution Time: 1.0 s Mode: Transmission.

Form C: X-ray Tube: Cu(1.54059 A) Voltage: 45 kV Amperage: 40 mA ScanRange: 1.00-39.99°2θ Step Size: 0.017°2θ Collection Time: 719 s ScanSpeed: 3.3°/min Slit: DS: 1/2° SS: null Revolution Time: 1.0 s Mode:Transmission

Differential Scanning Calorimetry (DSC)

DSC was performed using TA Instruments 2920 and Q2000 differentialscanning calorimeters. Temperature calibration was performed usingNIST-traceable indium metal. The sample was placed into an aluminum DSCpan, covered with a lid, and the weight was accurately recorded. Aweighed aluminum pan configured as the sample pan was placed on thereference side of the cell. Endotherm temperatures reported aretransition maxima unless specified. The data acquisition parameters andpan configuration for each thermogram are displayed in the image in theFigures Section. The method code on the thermogram is an abbreviationfor the start and end temperature as well as the heating rate; e.g.,−50-250-10 means “from −50° C. to 250° C., at 10° C./min”. The followingtable summarizes the abbreviations used in each image for panconfigurations:

Abbreviation (in comments) Meaning T0 Tzero, indicates pan has no lip CLid crimped MP manual pinhole

Thermogravimetric Analysis (TGA)

TG analyses were performed using a TA Instruments Q5000 IRthermogravimetric analyzer. Temperature calibration was performed usingnickel and Alumel. Each sample was placed in an aluminum pan. The samplewas hermetically sealed, the lid pierced, then inserted into the TGfurnace. The furnace was heated under nitrogen. The data acquisitionparameters for each thermogram are displayed in the image in the FiguresSection. The method code on the thermogram is an abbreviation for thestart and end temperature as well as the heating rate; e.g., 00-350-10means “from current temperature to 350° C., at 10° C./min”, that is thetemperature was not equilibrated prior to the start of the analysis.

Thermogravimetric Infrared (TG-IR) Spectroscopy

Thermogravimetric infrared (TG-IR) analysis was performed on a TAInstruments thermogravimetric (TG) analyzer model 2050 interfaced to aMagna-IR 560® Fourier transform infrared (IR) spectrophotometer (ThermoNicolet) equipped with an Ever-Glo mid/far IR source, a potassiumbromide (KBr) beamsplitter, and a mercury cadmium telluride (MCT-A)detector. The IR wavelength verification was performed usingpolystyrene, and the TG calibration standards were nickel and Alumel™.The sample was placed in a platinum sample pan, and the pan was insertedinto the TG furnace. The TG instrument was started first, immediatelyfollowed by the FT-IR instrument. The TG instrument was operated under aflow of helium at 90 and 10 cc/min for the purge and balance,respectively. The furnace was heated under helium at a rate of 20°C./minute to a final temperature of 97° C. IR spectra were collectedapproximately every 16 seconds for approximately 13 minutes. Each IRspectrum represents 16 co-added scans collected at a spectral resolutionof 4 cm⁻¹. Volatiles were identified from a search of the HighResolution Nicolet Vapor Phase spectral library (v. 1990-1994).

Hot Stage Microscopy

Hot stage microscopy was performed using a Linkam hot stage (FTIR 600)mounted on a Leica DM LP microscope equipped with a SPOT Insight™ colordigital camera. Temperature calibrations were performed using USPmelting point standards. Samples were placed on a cover glass, and asecond cover glass was placed on top of the sample. As the stage washeated, each sample was visually observed using a 20× objective withcrossed polarizers and a first order red compensator. Images werecaptured using SPOT software (v. 4.5.9).

Karl-Fischer Titration (KF)

Coulometric KF analysis for water determination was performed using aMettler Toledo DL39 KF titrator. A blank titration was carried out priorto analysis. The sample was prepared under a dry nitrogen atmosphere,where 11 to 78 mg of the sample was dissolved in approximately 1 mL dryHydranal-Coulomat AD in a pre-dried vial. The entire solution was addedto the KF coulometer through a septum and mixed for 10 seconds. Thesample was then titrated by means of a generator electrode, whichproduces iodine by electrochemical oxidation: 2 I⁻→I₂+2e⁻. Tworeplicates were obtained to ensure reproducibility.

Fourier Transform Infrared (IR) Spectroscopy

IR spectra were acquired on Nexus 670® IR spectrophotometer (ThermoNicolet) equipped with an Ever-Glo mid/far IR source, a potassiumbromide (KBr) beamsplitter and a deuterated triglycine sulfate (DTGS)detector. Wavelength verification was performed using NIST SRM 1921b(polystyrene). An attenuated total reflectance (ATR) accessory(Thunderdome™, Thermo Spectra-Tech), with a germanium (Ge) crystal wasused for data acquisition. Each spectrum represents 256 co-added scanscollected at a spectral resolution of 4 cm⁻¹. A background data set wasacquired with a clean Ge crystal. A Log 1/R (R=reflectance) spectrum wasobtained by taking a ratio of these two data sets against each other.

Fourier Transform Raman (Raman) Spectroscopy

Raman spectra were acquired on a Raman module interfaced to a Nexus 670IR spectrophotometer (Thermo Nicolet) equipped with an indium galliumarsenide (InGaAs) detector. Wavelength verification was performed usingsulfur and cyclohexane. Each sample was prepared for analysis by placingthe sample into a glass tube, capillary or pellet and positioning in agold-coated holder. Approximately 1 W of Nd:YVO₄ laser power (1064 nmexcitation wavelength) was used to irradiate the sample. The dataacquisition parameters for each spectrum are displayed above the imagein Appendix C

Solution Proton Nuclear Magnetic Resonance (¹H-NMR)

The ¹H-NMR spectra were acquired with a Varian ^(UNITY)INOVA-400spectrometer. The sample samples were prepared by dissolvingapproximately 3 to 13 mg of sample in DMSO-d₆ containing TMS. The dataacquisition parameters are displayed in the first plot of the spectrumin Appendix C.

Solid-state Carbon Nuclear Magnetic Resonance (¹³C-NMR)

The ¹³C-NMR solid-state NMR spectra were acquired with a Varian^(UNITY)INOVA-400 spectrometer. The samples were prepared by packingthem into 4 mm PENCIL type zirconia rotors and rotating at 12 kHz at themagic angle. The data acquisition parameters are displayed in the firstplot of the spectrum in Appendix C.

Dynamic Vapor Sorption (DVS)

DVS data were collected on a VTI SGA-100 Vapor Sorption Analyzer. NaCland PVP were used as calibration standards. Samples were not dried priorto analysis. Sorption and desorption data were collected over a rangefrom 5 to 95% RH (or 55 to 95% RH for Form B) at 10% RH increments undera nitrogen purge. The equilibrium criterion used for analysis was lessthan 0.0100% weight change in 5 minutes with a maximum equilibrationtime of 3 hours. Data were not corrected for the initial moisturecontent of the samples.

XRPD Indexing

XRPD patterns of treprostinil Forms A and B were indexed using X'PertHigh Score Plus [12]. Agreement between the allowed peak positions,marked with red bars, and the observed peaks indicates a consistent unitcell determination. Space groups consistent with the assigned extinctionsymbol, unit cell parameters, and derived quantities are tabulated belowthe figures. Indexing and structure refinement are computational studieswhich are performed under the “Procedures for SSCI Non-cGMP Activities.”

* * *

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention.All of the publications, patent applications and patents cited in thisspecification are incorporated herein by reference in their entirety.

What is claimed is:
 1. A treprostinil Form C having an X-ray powderdiffractogram comprising the following peak: 6.55 °2θ±0.2 °2 θ, asdetermined on a diffractometer using Cu-Kα radiation at a wavelength of1.54059 Å, in substantially pure form.
 2. The treprostinil Form C ofclaim 1, wherein the diffractogram is substantially as shown in FIG. 16.3. The treprostinil Form C of claim 1 having a purity of at least 90%aside from residual solvents.
 4. The treprostinil Form C of claim 3having the purity of at least 95% aside from the residual solvents. 5.The treprostinil Form C of claim 3 having the purity of at least 98%aside from the residual solvents.
 6. The treprostinil Form C of claim 3having the purity of at least 99% aside from the residual solvents. 7.The treprostinil Form C of claim 3 having the purity of at least 99.9%aside from the residual solvents.
 8. The treprostinil Form C of claim 3,wherein the purity is determined by NMR integration or by analyzing anX-ray powder diffractogram.
 9. The treprostinil Form C of claim 1 thatis substantially free of any other form of crystalline treprostinil. 10.The treprostinil Form C of claim 1, wherein a differential scanningcalorimetry (DSC) curve of Form C comprises a minor endotherm at about78.3° C. and a major endotherm at about 126.3° C.
 11. The treprostinilForm C of claim 10, wherein the DSC curve is substantially as shown inFIG.
 17. 12. The treprostinil Form C of claim 1 in an amount of 1 gramto 50 kg.
 13. A method of making treprostinil Form C of claim 1,comprising exposing treprostinil monohydrate Form A or treprostinilmonohydrate Form B to a reduced pressure and a temperature less than 42°C.
 14. A pharmaceutical formulation comprising treprostinil Form C ofclaim 1 and a pharmaceutically carrier or excipient.
 15. A solid dosageform for oral administration comprising treprostinil Form C of claim 1mixed with at least one inert pharmaceutical carrier.
 16. The soliddosage form of claim 15, which is a capsule, a tablet, a pill, a powderor a granule.
 17. A crystalline treprostinil monohydrate Form A, havingan X-ray powder diffractogram comprising the following peaks: 11.6,16.2, and 20.0 °2θ±0.2 °2θ, as determined on a diffractometer usingCu-Kα radiation at a wavelength of 1.54059 Å, in substantially pureform.
 18. The crystalline treprostinil monohydrate Form A of claim 17,wherein the diffractogram further comprises peaks at 5.2, 21.7, and 27.7°2θ±0.2 °2θ.
 19. The crystalline treprostinil monohydrate Form A ofclaim 17, wherein the diffractogram is as shown in FIG.
 2. 20. Thecrystalline treprostinil monohydrate Form A of claim 17, wherein thecrystalline treprostinil monohydrate form A has a differential scanningcalorimetry (DSC) curve that comprises a minor endotherm at about 78.3°C. and a major endotherm at about 126.3° C.
 21. The crystallinetreprostinil monohydrate Form A of claim 20, wherein the DSC curve is asshown in FIG.
 3. 22. The crystalline treprostinil monohydrate Form A ofclaim 17 having a purity of at least 90% aside from residual solvents.23. The crystalline treprostinil monohydrate Form A of claim 22 havingthe purity of at least 95% aside from the residual solvents.
 24. Thecrystalline treprostinil monohydrate Form A of claim 22 having thepurity of at least 98% aside from the residual solvents.
 25. Thecrystalline treprostinil monohydrate Form A of claim 22 having thepurity of at least 99% aside from the residual solvents.
 26. Thecrystalline treprostinil monohydrate Form A of claim 22 having thepurity of at least 99.9% aside from the residual solvents.
 27. Thecrystalline treprostinil monohydrate Form A of claim 22, wherein thepurity is determined by NMR integration or by analyzing an X-ray powderdiffractogram.
 28. The crystalline treprostinil monohydrate Form A ofclaim 17, wherein said Form A is free of any other form of crystallinetreprostinil.
 29. The crystalline treprostinil monohydrate Form A ofclaim 17 in an amount of 1 gram to 50 kg.
 30. A crystalline treprostinilmonohydrate Form B, having an X-ray powder diffractogram comprising thefollowing peaks: 5.9, 12.1, and 24.4 °2θ±0.2 °2θ, as determined on adiffractometer using Cu-Kα radiation at a wavelength of 1.54059 Å, insubstantially pure form.
 31. The crystalline treprostinil monohydrateForm B of claim 30, wherein the diffractogram further comprises peaks at10.7, 20.6, and 22.3°2θ±0.2°2θ.
 32. The crystalline treprostinilmonohydrate Form B of claim 30, wherein the diffractogram is as shown inFIG.
 9. 33. The crystalline treprostinil monohydrate Form B of claim 30,wherein the crystalline treprostinil monohydrate Form B has adifferential scanning calorimetry (DSC) curve that comprises a minorendotherm at about 74.8° C. and a major endotherm at about 125.2° C. 34.The crystalline treprostinil monohydrate Form B of claim 33, wherein theDSC curve is as shown in FIG.
 10. 35. The crystalline treprostinilmonohydrate Form B of claim 30 having a purity of at least 90% asidefrom residual solvents.
 36. The crystalline treprostinil monohydrateForm B of claim 35 having the purity of at least 95% aside from theresidual solvents.
 37. The crystalline treprostinil monohydrate Form Bof claim 35 having the purity of at least 98% aside from the residualsolvents.
 38. The crystalline treprostinil monohydrate Form B of claim35 having the purity of at least 99% aside from the residual solvents.39. The crystalline treprostinil monohydrate Form B of claim 35 havingthe purity of at least 99.9% aside from the residual solvents.
 40. Thecrystalline treprostinil monohydrate Form B of claim 35, wherein thepurity is determined by NMR integration or by analyzing an X-ray powderdiffractogram.
 41. The crystalline treprostinil monohydrate Form B ofclaim 30, wherein said Form B is free of any other form of crystallinetreprostinil.
 42. The crystalline treprostinil monohydrate Form B ofclaim 30 in an amount of 1 gram to 50 kg.